Journal of Reproduction and Development, Vol. 58, No 1, 2012 —Original Article— Upregulation of Interferon-stimulated Genes and Interleukin-10 in Peripheral Blood Immune Cells During Early Pregnancy in Dairy Cows Koumei SHIRAsuNA1), Haruka MATsumOTO1), Eiji KOBAYASHI1), Akane NiTTA1), Shingo HANedA2), Motozumi MATsui2), Chiho KAWASHimA3), Katsuya KidA3), Takashi SHimiZU1) and Akio MIYAMOTO1) 1)Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan 2)Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan 3)Field Center of Animal Science and Agriculture, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan Abstract. In cows, interferon-tau (IFNT) regulates maternal recognition around days 15–19 after artificial insemination (AI). The present study hypothesized that if key target genes of IFNT are clearly upregulated in earlier stages of pregnancy, these genes could be use as indices of future pregnancy in cows. Therefore, we determined the expression of these genes in peripheral blood mononuclear leukocytes (PBMCs) and polymorphonuclear granulocytes (PMNs) during the maternal recognition period (MRP). Twenty multiparous Holstein cows were subjected to AI on day 0 and categorized into the following groups: pregnancy (Preg, n = 9), embryonic death (ED, n = 5) and non-pregnancy (NP, n = 6). Progesterone levels in the Preg group were higher than those in the NP group on days 12–21. ISG15 and OAS-1 (IFN-stimulated genes: ISGs) mRNA in PBMCs on day 8 was higher in the Preg group than in the NP group, and these mRNAs in PMNs was higher in the Preg group on day 5 than in the NP and ED groups. Interleukin-10 (IL-10, Th2 cytokine) mRNA expression increased on day 8 in the PBMCs of pregnant cows. Tumor necrosis factor α (TNFα, Th1 cytokine) mRNA expression was stable in all groups. In an in vitro cell culture experiment, IFNT stimulated mRNA expression of ISGs in both PBMCs and PMNs. IFNT stimulated IL-10 mRNA expression in PBMCs, whereas IFNT increased TNFα mRNA levels in PBMCs in vitro. The results suggest that ISGs and IL-10 could be responsive to IFNT before the MRP in peripheral blood immune cells and may be useful target genes for reliable indices of pregnancy before the MRP. Key words: Cow, Immune cells, Interferon tau, Pregnancy (J. Reprod. Dev. 58: 84–90, 2012) uring the past 5 decades, milk production per cow has dramati- in nonpregnant cows on days 18 and 20 after artificial insemination Dcally increased because of improved management, nutrition, (AI) [7–9]. Technology for the early detection of pregnancy is needed and genetic selection [1, 2]. In contrast, the decreases in fertility to identify nonpregnant cows and to synchronize and artificially and conception rates of the modern high-producing dairy cow are inseminate these cows prior to the next ovulation, and many stud- the major causes of economic loss for dairy producers [2, 3]. After ies have focused on identifying easy-to-use markers of pregnancy establishment of pregnancy in domestic ruminants, the conceptus using blood samples. These previous studies investigated possible secretes interferon-tau (IFNT) as a maternal recognition factor marker genes using whole-blood leukocytes and peripheral blood [4]. IFNT acts in the uterus around day 16 after insemination and mononuclear cells (PBMCs) because leukocytes and PBMCs contain prevents luteolysis by inhibiting prostaglandin F2α release, resulting T lymphocytes, which are required to induce an immune response. in the maintenance of corpus luteum function; therefore, this period However, leukocytes comprise different types of cells such as T is termed the maternal recognition period (MRP) [5]. IFNT induces lymphocytes, monocytes, neutrophils and eosinophils. Thus, in the the synthesis and secretion of IFN-stimulated genes (ISGs) such present study, we investigated the gene expression of ISGs in PBMCs as ISG15, 2′,5′-oligoadenylate synthetase (OAS-1), IFN regulatory (lymphocytes and monocytes) and polymorphonuclear granulocytes factor 1, Mx1 and Mx2 not only in the uterus but also in blood (PMNs: neutrophils and eosinophils) separately following AI. cells in ewes and cows [6–10]. In fact, in bovine peripheral blood Peak levels of IFNT are observed around days 15–19 of preg- leukocytes, ISG15 mRNA levels were higher in pregnant cows than nancy in cows. However, Green et al. [9] reported that Mx2 mRNA levels in leukocytes increased on day 14 of pregnancy following AI compared with the levels in nonpregnant cows. Additionally, IFNT Received: June 27, 2011 mRNA is first expressed in 16-cell stage in vitro fertilized embryos Accepted: October 1, 2011 on day 4 [11], and IFNT protein was detected in the trophectoderm Published online in J-STAGE: November 4, 2011 ©2012 by the Society for Reproduction and Development of nonhatched blastocysts on day 7 using confocal microscopy [12]. Correspondence: A Miyamoto (e-mail: [email protected]) These findings suggest that IFNT signals are transmitted before ISGS AND IL-10 IN IMMUNE CELLS DURIGN EARLY PREGNANCY 85 the MRP (around days 15–19) in cows. On the other hand, general In vitro study alterations occur in the maternal inflammatory and immune system PBMCs and PMNs were isolated from whole blood collected via during pregnancy. A shift from T-helper 1 (Th1) to T-helper 2 (Th2) jugular venipuncture on days 4–5, 7–8 and 10–12 of the estrous cytokines is considered responsible for this immunomodulation cycle (day of ovulation = day 1; non-AI cows, n = 4) as described during pregnancy. For example, inflammatory cytokines such as previously [17, 18]. Isolated PBMCs and PMNs were resuspended at tumor necrosis factor α (TNFα) and IFNγ can terminate normal a concentration of 5 × 106 cell/ml in RPMI 1640 medium contain- pregnancy upon injection during early gestation [13]. In contrast, ing 0.1% FBS, gentamicin (50 mg/l) and amphotericin B (2.5 mg/l) anti-inflammatory cytokines such as interleukin-10 (IL-10) and purchased from Sigma (St. Louis, MO, USA). Cells were incubated in transforming growth factor β (TGFβ) play crucial roles in prevent- the same medium with the following treatments: control (no further ing fetal loss [13, 14]. Therefore, we hypothesized that IFNT can addition) or IFNT (0.1, 1 or 10 ng/ml; recombinant bovine IFNT potentially regulate ISGs and the Th1/Th2 balance in immune was purchased from Pestka Biomedical Laboratories, Piscataway, cells during the early phase of pregnancy in cows because IFNT NJ, USA. Specific activity as determined in a viral resistance as- prevents fetal demise in pregnant mice [13]. say using bovine kidney MDBK cells was 1 × 106 IU/ml). Each The aim of the present study was to examine the time-dependent treatment was performed in 48-well plates in duplicate. Incubation changes in the mRNA expression of ISGs and Th1/Th2-related fac- was performed for 4 h at 37 C. At the end of the treatment period, tors in PBMCs and PMNs after AI in vivo and the effect of IFNT cells were stored at −80 C until mRNA expression was analyzed. treatment in vitro on PBMCs and PMNs obtained on different days of the estrous cycle. RNA isolation and real-time PCR Total RNA was extracted following the protocol of Chomczynski Materials and Methods and Sacchi using TRIzol reagent [19] as in our previous study [20]. The extracted total RNA was stored in RNA storage solu- In vivo study tion (Ambion, Austin, TX, USA) at –80 C until use for cDNA The blood collection experiments were conducted at the Field production. RNA samples were treated with DNase using an RQ1 Center of Animal Science and Agriculture, Obihiro University, RNase-Free DNase kit (Promega, Madison, WI, USA) as in our and all experimental procedures complied with the Guidelines for previous study [20]. The synthesized cDNA was stored at –30 C. the Care and Use of Agricultural Animals of Obihiro University. The mRNA expression of ISG15, OAS-1, IL-10, TNFα, and Between September 2010 and December 2010, 20 multiparous β-actin was quantified by real-time PCR with a LightCycler (Roche Holstein cows were artificially inseminated on day 0. Blood was Diagnostics GmbH, Mannheim, Germany) as described in our collected on days 1–2, 5, 8, 12, 15, 18, and 21 of pregnancy fol- previous study [20]. The primers used for real-time PCR were as lowing AI to detect plasma progesterone (P4) concentrations and follows: 5′-ggtatgatgcgagctgaagcactt-3′, forward, and 5′-acctccct- separate immune cells. In addition, blood was continuously col- gctgtcaaggt-3′, reverse, for ISG15 (Accession No. NM_174366); lected on days 24, 27, 30, 35, and 40 of pregnancy to detect plasma 5′-taggcctggaacatcaggtc-3′, forward, and 5′-tttggtctggctggattacc-3′, P4 concentrations. Pregnancy was confirmed by ultrasonography reverse, for OAS-1 (Accession No. MN_001040606); 5′-ttctgccct- on day 40. Plasma P4 concentrations were determined by direct gcgaaaacaa-3′, forward, and 5′-tctcttggagctcactgaagactct-3′, re- enzyme immunoassays [15]. verse, for IL-10 (Accession No. NM_174088); 5′-tgacgggctttacct- Whole blood (20 ml) was mixed with an equal volume of PBS. catct-3′, forward, and 5′-tgatggcagacaggatgttg-3′, reverse, for TNFα The suspension was layered onto Ficoll-Paque solution (Lymphoprep, (Accession No. AF_348421); and 5′-ccaaggccaaccgtgagaaaat-3′, Axis-Shield, Oslo, Norway) and then centrifuged at 1000 × g at forward, and 5′-ccacattccgtgaggatcttca-3′, reverse, for β-actin 10 C for 30 min as described previous study [16–18]. The plasma (Accession No. MN_173979.3). The PCR products were subjected and buffy coat (population of mononuclear cells) were separated to electrophoresis, and the target band was excised and purified as PBMCs layer. After removing PBMCs, contaminating red blood using a DNA purification kit (SUPRECTM-01; Takara Bio, Otsu, cells were washed in hypotonic distilled water for approximately 10 Japan).